The present invention relates to a method for isolating RNA from a sample and to a kit for use in such a method.
The process by which RNA is separated from other molecules, in particular other cellular components such as proteins, carbohydrates, lipids and DNA is widely known and described in the literature. It is a key process in the understanding of gene function and structure and drug development. Furthermore nucleic acid based diagnostic procedures for detecting RNA viruses such as HCV and HIV absolutely require the viral genomic RNA to be isolated in an intact and relatively pure form. In order to separate RNA in an intact and pure form from other biological material it is necessary to rapidly inactivate all ribonucleases that are present and separate the RNA based on a biophysical property unique to the RNA. However, due to the similar physical characteristics of RNA and DNA, RNA preparations are frequently if not always contaminated with DNA, leading to major difficulties in the analysis of results.
Currently mRNA is separated from genomic DNA by using a method based on oligo (dT) interacting with the poly A tail (Aviv and Leder., Proc. Natl. Acad. Sci. USA. 69, 1408-1412 (1972). However, A-rich DNA sequences are co-purified with this method leading to mRNA contaminated with A-rich DNA. Alternatively, nucleases that are specific for either RNA or DNA can be employed. Such highly purified enzymes are costly to use and frequently require removal before the nucleic acid can be used. For example RNase free, DNase must be removed by phenol extraction or heat inactivation otherwise it will destroy for example PCR primers or other DNA based reagents in all post-nuclease reactions. Another method is to use a mixture of phenol, chloroform, isoamyl alcohol (50:49:1); whereby DNA preferentially partitions into the organic phase whilst RNA remains in the aqueous phase. This method at best still leaves a significant amount of DNA contaminating the RNA and hence is of limited practical use. Another method is the TRI REAGENT(trademark) (Molecular Research Centre, Inc) that allows the simultaneous separation of DNA, RNA and proteins. However, it requires careful separation of different phases from each other and subsequent centrifugation steps whilst not assuring complete separation of DNA from the RNA due to the difficulty of pipetting small volumes of liquid without cross-contamination. Another method is anion exchange chromatography which can separate RNA from DNA but the expense and difficulty of setting up the column as well as the restriction to purifying only small nucleic acids precludes its use from the majority of laboratories. Numerous methods to purify RNA are described in general texts (Jones et al., (1994) in RNA Isolation and Analysis. Bios. Oxford., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, CSH.).
The present invention provides a preparative method for isolating RNA comprising an oligo- or polynucleotide from a sample, which method comprises:
(a) treating the sample with a reactant capable of covalently modifying the 2xe2x80x2-OH position of the ribose rings of the RNA under conditions so that a proportion of the 2xe2x80x2-OH positions of the ribose rings bear a substituent; and
(b) preparing isolated RNA therefrom by separating material containing the substituent from the sample on the basis of a property of the substituent.
The RNA may be mRNA, tRNA, rRNA, viral RNA, viroid RNA, synthetic RNA such as chemically synthesised or in vitro transcribed forms, or any other form of RNA, such as hnRNA. The RNA may be a mixture of different types of RNA and may be in single- or double-stranded form, linear or circular and contain internal regions of secondary and tertiary structure such as is commonly found in tRNA. According to the present invention an oligonucleotide generally has a sequence of up to about 80 bases and a polynucleotide generally has a sequence length of more than about 80, preferably more than about 100 bases. A preferred length for a polynucleotide is at least 1000 bases.
The mRNA may or may not have a cap and/or poly A tail. The RNA used in the present invention is preferably naturally-occurring. A naturally-occurring RNA according to the present invention typically comprises a nucleotide sequence which is found in nature and which has a structural function or generally encodes a polypeptide having biological activity, or such a nucleotide sequence which is modified, for example to alter in some way the biological activity of the polypeptide encoded thereby. Whilst the naturally-occurring RNA is preferably obtained by transcription from a suitable RNA or DNA template, itself usually naturally-occurring, in some cases the naturally-occurring RNA can be obtained synthetically. RNA according to the present invention does not encompass simple homopolynucleotides (poly A, poly U, poly G and poly C) which can be generated synthetically but are biologically non-functional.
The naturally occurring RNA can be derived from a biological material such as bacteria, viruses such as those causing infection in humans, animals or plants, viroids, or cells such as fungal, animal and plant cells.
An important aspect of this invention is modification of mRNA, rRNA and viral RNA since they are of major scientific and clinical interest and serve as a good example of the problems encountered when manipulating RNA. The invention further provides methods for obtaining intact full-length copies of mRNA, rRNA, viral RNA and other types of RNA isolated from cellular sources or extracellular fluids that demonstrate increased stability in conditions that would otherwise destroy a major fraction of the unmodified RNA.
Measuring the Percentage Modification of RNA
Due to the polymeric nature of RNA, it is difficult to measure its molecular weight above 100 nucleotides using mass spectrometry because a large amount of RNA degradation occurs during the analytical process. However, RNA (tRNA) up to 142 nucleotides (Nordhoff et al., (1993) Nucleic Acids Res. 21:3347; Gruic-Sovulj et al., (1997) Nucleic Acids Res. 25:1859; Tolson and Nicholson (1998) Nucleic Acids Res. 26:446) and double stranded DNA up to 500 base-pairs (Bai et al. (1995) Rapid Comm. Mass Spectrom. 9:1172; Taranenko et al., (1998) Nucleic Acids Res. 26:2488; Ausdall and Marshall (1998) Anal. Biochem. 256:220) have been measured using MALDI mass spectrometry (for reviews see; Smith (1996) Nat. Biotech. 14:1084; Murray (1996) J. of Mass Spectrom. 31:1203. Phosphate (Schuette et al., (1995) J. Pharm. Biomed. Anal. 13:1195; Sinha et al., (1994) Nucleic Acids Res. 22:3119) and chemically modified oligonucleotides (Potier et al., (1994) Nucleic Acids Res. 22:3895) have also been measured using mass spectrometry.
Although there is a molecular weight limitation to a few hundreds of nucleotides when using mass spectrometry, it provides a simple, automated means to accurately determine the exact molecular weight and therefore the percentage modification of a polynucleotide. Optimisation relies on a number of factors such as the type of mass spectrometry being carried out (electro-spray, MALDI-TOF etc), the method used to purify the modified RNA from the modification reaction, the size of the polynucleotide, the ionisation matrix used, the method used to remove cations from the RNA, positive or negative ion mode and the voltage strength used (Fenn et al., 1989) Science 246:64). Capillary high performance liquid chromatography can be used prior to mass spectrometry of RNA because desalting and other purification steps are not required prior to ionisation (Taniguchi and Hayashi (1998) Nucleic Acids Res. 26:1481).
To measure the molecular weight and hence the percentage modification of polynucleotides consisting of thousands of nucleotides requires a different approach. In certain situations where it is preferable to measure the percentage modification of the polynucleotide using more precise means a degradative step may be employed followed by an analytical process. It is expected that degradation of the modified polynucleotide using chemical or enzymatic means will, depending on the method used leave the 2xe2x80x2-OH modification attached to the ribose sugar allowing the amount of modification to be established by mass spectrometry or high performance liquid chromatography (HPLC). HPLC and gas chromatography analysis of nucleotides has been described (Gehrke and Patel (1077) J. Chromat. 130:103; Iwase et al., (1975) J. Chromat. 106:213; Kemp et al., (1982) J. Chromat. 241:325).
In order to establish the percentage of nucleotides that are modified, degradation of the polynucleotide should follow the modification reaction. Methods have been described for enzymatic cleavage methods employing ribonucleases RNase T1, RNase A, RNase U2, RNase PhyM, RNase CL3, nuclease S7 and cusativin, chemical cleavage methods using sulfuric acid (Jones et al., (1994) RNA Isolation and Analysis, chapter 3, Bios Scientific Publishers, Oxford) and physical methods using post source decay (Hahner et al., (1997) Nucleic Acids Res. 25:1957; Taniguchi and Hayashi (1998) Nucleic Acids Res. 26:1481; Kirpekar et al., (2000) RNA 6:296).
It will be understood that the 2xe2x80x2-OH modification may inhibit degradation of the polynucleotide. However, by empirically determining the sensitivity of the modified RNA to a range of conditions it will be should be possible in most cases to select conditions that are suitable for chain cleavage. For example, it has been found that acetylated RNA is readily cleaved by nuclease Bal 31. Whilst alkali cleaves acetylated RNA it also results in acetyl cleavage so unless the amount of cleaved acetyl groups is measured by mass spectrometry, acetylated nucleotides will not be detected. For example, acid cleavage of the modified polynucleotide can be used for base sensitive modifications, whilst base cleavage can be used for acid sensitive modifications. It will also be understood that other degradation products such as dinucleotides, trinucleotides etc will also be suitable for measuring the percentage modification of the polynucleotide. Whether it is the nucleotide, dinucleotide or larger fragments that are being measured, in each case it is the ratio of the number of fragments bearing a modification compared with the number of fragments not bearing a modification that provides the percentage modification.
Other methods that are capable of measuring high molecular weight RNA such as analytical ultracentrifugation to find the sedimentation coefficients (Svedberg units) are imprecise, require large amounts of starting material and are dependent on the conformation of the RNA (for review, see Jones et al., (1994) RNA Isolation and Analysis, chapter 3, Bios Scientific Publishers, Oxford). Despite these drawbacks, analytical ultracentrifugation using denaturing sucrose or isokinetic gradients may be useful to measure very large molecular weight changes in abundant RNA samples.
It is now much more common to measure the molecular weight of polynucleotides using electrophoretic separation in polyacrylamide or agarose gels. Detailed descriptions of the preparation, use and handling of electrophoresis gels is described in several publications (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, CSH; Jones (1995) Gel Electrophoresis: Nucleic Acids Essential Techniques, Wiley). Denaturing gels are preferred to non-denaturing gels because they reduce conformational effects providing a means to measure the true molecular weight of the linear polynucleotide (Jones (1995) Gel Electrophoresis: Nucleic Acids Essential Techniques, page 47, Wiley). There are a variety of denaturants that can be used such as DMSO (50-90%), glyoxal (10-30%), formaldehyde (3% w/v), formamide (50-98%), heat (60-80xc2x0 C.), methyl mercuric hydroxide (3-5 mM), sodium iodoacetate (10 mM), 2-pyrrolidone (5%) and urea (6-8 mM). It is known that incomplete denaturation of the polynucleotide leads to anomalous migration so that more than one denaturing condition may be required such as 8 M urea plus 5% pyrrolidonone or 8 M urea run at 60xc2x0 C. (Rosenblum et al., (1997) Nucleic Acids. Res. 25:3925) Capillary electrophoresis provides an excellent means to carry out such molecular weight determinations and suitable methods have been described for RNA (Engel and Dieguez-Lucena (1993) Nucleic Acids Res. 21:759).
Comparative measurements of polynucleotide migration between different gels is difficult because the distance migrated is dependent on the buffer used, gel concentration and temperature. Therefore it is preferred that comparisons be made with both molecular weight standards and sample polynucleotides in the same gel. It is also known that certain percentages of the sieving matrix such as polyacrylamide or agarose are optimal for certain lengths of nucleic acids and above a certain percentage of acrylamide or length of polynucleotide (the exclusion limit) separation as a function of length does not occur. Therefore, measurements of the molecular weight should be made within such known limits (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, CSH; Jones (1995) Gel Electrophoresis: Nucleic Acids Essential Techniques, Wiley).
It has been found that using a 20 cm 6 M urea 5% polyacrylamide gel that a 250 nucleotide acetylated RNA runs approximately 20 mm apart from the non-modified form as a tight band. Therefore, measuring the amount of modification of smaller modifying groups than acetyl (42 daltons) should be feasible. The acetylated RNA also runs at a position predicted to be 100% modified as compared with RNA size markers.
It is common practice to be able to separate in a denaturing sequencing gel DNA polynucleotides differing by as little as 1 nucleotide in a total length of 500 nucleotides i.e. by 0.2% or less difference in length (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, CSH.). It is therefore reasonable to expect that accurate measurements can be made for the molecular weight of RNA polynucleotides in their modified and unmodified forms when the modifying group is large, for example 28 daltons for formyl and 42 daltons for acetyl.
Measuring the percentage modification with other modifying groups may also be possible providing that the molecular mass increase as a result of the modification is sufficient. For example, halogenation of the 2xe2x80x2-OH group should be readily measured for chlorine (35.4 daltons) and bromine (79.9 daltons) substitution of the 2xe2x80x2-OH group.
Calculating the percentage modification may be carried out by measuring the migration of known RNA size markers in a gel such as a 6 M urea-5% acrylamide sequencing gel and plotting migration (mm) versus molecular weight (daltons) to obtain a standard curve. Because the exact, molecular weights of all the markers are known, it is straight forward to graph the relationship between the mobility in the gel versus the known molecular weight of each marker. The percentage modification for an RNA of known length with a known mass for the modifying group can then be readily calculated by comparison with this standard curve.
An alternative means to determine the percentage modification is to use a radioactively labelled reactant such as 14 C or 3 H acetic anhydride to modify RNA and then determining the molar ratio of radioactive acetyl groups to nucleotides in a known amount of RNA sample. If the molar ratio is 1:1, then 100% of the 2xe2x80x2-OH groups are modified. It will be understood that radioactive isotopes can be incorporated into a wide variety of reagents.
Regiospecificity of the reaction can be determined by subjecting an identical sequence of DNA (or preferably single stranded DNA bearing uracil as a replacement to thymine), to identical reaction conditions as used for RNA. It is expected that the DNA is not substantially modified as measured by incorporation of radioactivity, gel electrophoresis mobility, mass spectrometry, HPLC or any other analytical means used if the reaction is regiospecific for the 2xe2x80x2-OH group.
Modification at the 2xe2x80x2-OH position is preferably substantially regiospecific. Thus, there is preferably substantially no modification of the bases, phosphodiester bonds and/or any other position within the RNA chain. In this way, the polynucleotide retains important properties of the RNA. For example, advantageously, the polynucleotide is preferably modified so that a single strand of the polynucleotide is replicable by a nucleic acid polymerase to generate a second strand of polynucleotide complementary to the single strand.
The modification at the 2xe2x80x2-OH position may be such that the entire OH of the 2xe2x80x2C of the ribose ring is replaced by a reactant group R as in 2xe2x80x2-R or by OR having 2xe2x80x2-OR where the xe2x80x94Oxe2x80x94 group may or may not originate from the 2xe2x80x2-OH group. Accordingly, the substituent at the 2xe2x80x2-OH position in this case is R or OR respectively.
Various reactants or reactant combinations may be used, optionally in the presence of a catalyst, to provide these substituents, as described in further detail in the Examples below. Advantageously, the reactant comprises an acid anhydride, an acid halide, an acyl cyanide, a carboxylic acid or an N-acylimidazole, an alkoxyalkyl halide, an alkylthioalkyl halide, an alkoxyalkoxyalkyl halide, a trialkylsilane halide or a trialkylsilane imidazole. Each of these reactants participates in a modification (e.g. acylation, modification with an ether-containing group or with a silane) reaction with the RNA. Preferably, the reactant is attached to a solid phase such as with benzoyl chloride polymer bound beads (BCPB) so that the modification reaction at the 2xe2x80x2-OH group covalently links the RNA to the solid phase. Under these reaction conditions, the reaction medium may further comprise an acylation catalyst. For example, where the reactant comprises an acid anhydride, this may be reacted with the RNA in the presence of a catalyst such as a fluoride ion or amino pyridine. As a further example, where the reactant comprises an acid chloride or N-acylimidazole, the reactant may be reacted with the RNA in the presence of an amino pyridine. As a further example, where the reactant comprises a carboxylic acid, this may be reacted with the RNA in the presence of a dehydrating agent or a catalyst, such as an isocyanide catalyst. A preferred aminopyridine catalyst is dimethyl aminopyridine (DMAP).
The organic solvent used in the reaction medium of the present invention may comprise an organic base and may comprise an organic solvent in which is dissolved the organic base or, in another embodiment, may be the organic base itself. It is preferred that the reactant is soluble in the organic solvent. In a preferred embodiment the reaction medium further comprises water. In this way RNA to be modified may be conveniently added to the organic solvent as an aqueous solution of RNA. Typical organic solvents include alkanes such as hexane and pentane, pyridine, acetonitrile, dimethylformamide, dichloromethane, acetone, diethyl ether, benzene, chloroform, ethyl acetate, light petroleum, tetrahydrofuran, carbon tetrachloride, dichloroethane, dioxane, carbon disulphide, nitromethane, dimethyl sulphoxide, hexamethylphosphoric triamide and toluene. Typical organic bases include pyridine, triethylamine, trimethylamine, diisopropylethylamine, N,N-diethylaniline, N,N-dimethylaniline, 1,5-diazabicyclo (4,3,0) non-5-ene (DBN), 1,8-diazabicyclo (5,4,0) undec-7-ene (DBU) and N-methylmorpholine. Triethylamine (CH3CH2)3N is a stronger (non-aqueous base) amine base than pyridine, aniline, diethylamine or trimethylamine but less so than pyrrolidone. It is one of the strongest amine bases. A preferred organic base which acts as an solvent is triethylamine (TEA). Where a catalyst is to be used, it is convenient for the catalyst to be soluble in the organic solvent as well. The water and the organic solvent may form different phases in the reaction medium. For example, the water and the organic solvent may be immiscible with one another and form phases which will separate upon standing. Where there is more than one phase, the RNA may be reacted with the reactant under conditions of phase transfer catalysis.
The amounts of water and organic solvent may be varied and will depend to some extent upon the particular organic solvent/base/catalyst system to be used. Advantageously, the reaction medium comprises at least 50% organic solvent, preferably at least 80%, more preferably at least 90% and more preferably at least 95% v/v. Typically, the ratio of water:organic solvent is in the range 1:50 to 1:10, preferably around 1:20.
It should be noted that if the percentage of 2xe2x80x2-OH groups modified is low, then the remaining sites in the chain bearing non-modified 2xe2x80x2-OH groups will be sensitive to degradation. Degradation may be caused during the reaction if basic solvents such as triethylamine, ethyldiisopropylamine or pyridine are used. Low percentage modification may be caused for a variety of reasons. Bulky modification reagents such as pentanoic anhydride react more slowly than smaller reactants such as propanoic anhydride, possibly because of steric hindrance between the RNA polynucleotide and the reactant. It would be expected that even larger reactants such as octanoic anhydride or oleic anhydride or those reactants that react relatively slowly would also lead to partial 2xe2x80x2-OH modification. In some cases, partial 2xe2x80x2-OH modification is desired because the RNA retains many of its original biophysical properties. Partial 2xe2x80x2-OH modification can be favoured over complete modification of the RNA by using large and bulky reactants, increasing the amount of RNA added, adding a competing reactive group, removing the catalyst, reducing the temperature and/or time of reaction or the amount of reactant present.
When the reactant itself is immobilised such as with benzoyl chloride polymer bound (BCPB) beads, it is unlikely that all the 2xe2x80x2-OH groups of a RNA polynucleotide will be modified because the RNA will be spatially constrained thereby restricting the other 2xe2x80x2-OH groups from contacting the other immobilised reactants. When partial 2xe2x80x2-OH modification is unavoidable or even desired, it is preferable to use reaction conditions that do not lead to RNA cleavage at non-modified sites such as by using non-basic solvents. It has been found that RNA in an intact form can be immobilised on BCPB beads when non-basic solvents such as tetrahydrofuran or dimethyl sulphoxide are used. If further protection/modification is desired, a second modifying reagent can be added during or subsequent to the first reaction thereby reacting and protecting the remaining 2xe2x80x2-OH groups. Alternatively, the secondary reactant could carry a label such as a fluorescent group (for example isatoic anhydride) allowing a simple means to quantitate the amount of RNA bound to the bead by measuring the amount of label subsequently attached to the RNA-beads. An example of such a secondary modification is given in the Examples.
In the absence of a catalyst, the reaction time is generally from 20 to 60 mins. In the presence of the catalyst, the reaction proceeds more quickly, the reaction time generally being completed within about 20 seconds.
On a vol/vol basis it is found that the ratio of reactant to reaction medium (especially acetic anhydride triethylamine/DMAP) is preferably in the range 1:200 to 1:10, more preferably around 1:20. Too little reactant gives a partial reaction and too much makes the reaction difficult to control.
In accordance with the method of the present invention, RNA is isolated from the sample by separating material which contains the substituent which has been used to modify the 2xe2x80x2-OH position of the ribose rings. Accordingly, a substituent needs to be selected so as to confer upon the RNA a property which the unmodified RNA does not possess. In one aspect, the substituent comprises an affinant capable of being recognised or bound by a partner such as a partner immobilised to a solid phase, or a protein. The affinant may be a hapten such as biotin or fluorescein which could be recognised and bound by an antibody or another protein such as streptavidin. Another useful affinant comprises a primary amino group capable of chemically reacting with an immobilised partner such as succinimide.
In one arrangement, the substituent comprises a hydrophobic substituent so that the RNA may be modified to render it more hydrophobic for the purpose of isolation. Both DNA and RNA are relatively hydrophilic molecules. It is quite difficult to separate RNA from DNA because their physical characteristics are similar. By increasing the hydrophobicity of RNA relative to DNA it is possible to improve the separation of the two types of nucleic acid. This is useful for either removing contaminating RNA from a DNA sample or removing contaminating DNA from RNA. For example it is important to remove bacterial RNA from a plasmid preparation prior to restriction enzyme analysis so that small DNA fragments are not obscured by the co-migrating RNA during agarose gel electrophoresis. Alternatively it is important to remove traces of DNA such as genomic DNA or viral DNA from cellular or viral RNA. Such DNA contamination often leads to false positives following RT-PCR amplification.
The hydrophobic substituent typically comprises a substituent, OR, wherein R comprises C1-C36 alkyl; C1-C36 alkenyl; C1-C36 alkynyl; C1-C36 haloalkyl; C1-C36 aminoalkyl; C1-C36 alkoxyalkyl; C1-C36 alkylthioalkyl; C1-C36 alkoxyalkoxyalkyl; C1-C36 haloalkoxyalkyl; C1-C36 aminoalkoxyalkyl; C6-C36 aryl; C6-C36 alkylaryl; C6-C36 arylalkyl; C6-C36 arylalkenyl; C1-C36 alkanoyl; C1-C36 alkenoyl; C1-C36 haloalkenoyl; C1-C36 haloalkanoyl; C2-C36 haloformylalkanoyl; C1-C36 C1-C36 aminoalkanoyl; C1-C36 azidoalkanoyl; C1-C36 carboxyalkanoyl; C1-C36 carboxyalkenoyl; C1-C36 carboxyalkynoyl; C1-C36 alkylaminoarylalkanoyl; C1-C36 alkoxycarbonyl; C1-C36 alkenyloxycarbonyl; C1-C36 alkylsulfonyl; C6-C36 arylalkanoyl; C6-C36 arylalkenoyl; C6-C36 aryloxyalkanoyl; C6-C36 alkylarylalkanoyl; C6-C36 haloarylalkanoyl; C6-C36 aminoarylalkanoyl; C1-C36 alkylsilanyl; C1-C36 trialkylsilanyl or C12-C28 diarylphosphano; or a substituent Rxe2x80x2, wherein Rxe2x80x2 comprises C1-C36 alkyl; C1-C36 alkenyl; C1-C36 alkynyl; C1-C36 haloalkyl; C1-C36 aminoalkyl; halo; amino; C1-C36 alkylamino; C6-C36 aryl; C1-C36 alkylaryl or C1-C36 arylalkyl.
In one embodiment, the hydrophobic substituent comprises a C4-C7 carbon chain or ring, preferably one which arises where the reactant comprises butyric anhydride, pentanoic anhydride, hexanoic anhydride or benzoic anhydride. In this case, it is preferred that the proportion of 2xe2x80x2-OH groups bearing the substituent is at least 10%.
Alternatively, the hydrophobic substituent may comprise a C8-C12 carbon chain or ring in which case it is preferred that the proportion of 2xe2x80x2-OH positions bearing the substituent is in the range 1 to 10%. As a further alternative, the hydrophobic substituent may comprise a C12-C36 carbon chain or ring more preferably a C12 to C24 carbon chain or ring. In this case, it is preferred that the proportion of 2xe2x80x2-OH positions bearing the substituent is up to 1%. Thus, whilst modification of up to 1% may provide a sufficiently hydrophobic modified RNA where a long chain length of substituent is used, substituents of lower carbon chain length require a higher percentage, perhaps in the range up to 95%, to be effective.
A number of methods for isolating RNA from samples treated in accordance with the present invention exist. For example, the step (b) of separating material containing the substituent from the sample may comprise contacting the treated sample from step (a) with a hydrophobic solid phase so as to bind the material containing the hydrophobic substituent and optionally washing the material bound to the hydrophobic solid phase. The hydrophobic solid phase may comprise hydrophobic particles. This method may further comprise a step of eluting the material bound to the hydrophobic solid phase by treating with a detergent, a chaotrope or a solvent, by lowering the salt concentration or by cleaving the substituent from the 2xe2x80x2-OH position of the ribose rings.
In a further embodiment, step (b) comprises treating the treated sample from step (a) with a lyotrophic salt to aggregate the material containing the hydrophobic substituent as an RNA precipitate, and isolating the precipitate. The lyotrophic salt preferably comprises ammonium sulphate, an alkali metal chloride, magnesium chloride or calcium chloride.
In a further embodiment, step (b) may comprise treating the treated sample with a non-polar solvent to form the hydrophobic liquid phase which contains the material containing the hydrophobic substituent, and isolating the hydrophobic liquid phase. The non-polar solvent typically comprises pentane, cyclohexane, toluene, benzene, light petroleum, xylene or hexane.
In a further embodiment, step (b) may comprise use of a membrane, such as a PVDF membrane, to separate the RNA. The addition of hydrocarbon chains to the 2xe2x80x2-OH position of RNA increases its hydrophobicity, that is its ability to interact with a hydrophobic surface. The use of hydrophilic PVDF filters to purify RNA has already been described in U.S. Pat. No. 5,958,677 (September 1999). However, this describes only the binding of RNA, which is in its unaltered form is a weakly hydrophobic molecule. Modified RNA molecules of increasing hydrophobicity can be created, as discussed above, by adding acyl groups of increasing chain length. Hydrophobicity increases in the order: 2xe2x80x2-OH less than formyl (xe2x80x94C(O)H) less than acetyl (xe2x80x94C(O)CH3) less than propanoyl (xe2x80x94C(O)CH2CH3) less than butanoyl (xe2x80x94C(O)CH2CH2CH3), i.e. according to the carbon chain length. Longer chain lengths than butanoyl were not tested but would be expected to follow the same trend. The increase in the strength of the hydrophobic interaction was tested by binding RNA and modified RNA to a polyvinylidene fluoride (PVDF) membrane and subjecting the bound material to increasingly stringent wash conditions (see Examples).
The reagent modifying the RNA 2xe2x80x2-OH group may be either free in solution or attached to a solid phase. In a particularly preferred aspect of this invention, a solid phase is employed. In this embodiment, the solid phase is not the same as the hydrophobic solid phase described above and is used as a reactant so that the solid phase itself is introduced as the substituent modifying the RNA (note the hydrophobic solid phase is employed only after the RNA is modified with a hydrophobic substituent). When using a solid phase, the modification reaction will lead to the covalent attachment of the RNA to the solid phase. This provides a convenient means to immobilise RNA to a solid phase in order to purify or analyse it. For example, RNA will be preferentially bound to the solid phase from a RNA, DNA and protein mixture such as a cell lysate or serum.
Advantages of using RNA covalently bound to a solid phase include ease of manipulation, purification, automation and compatibility with many important life science applications such as diagnostics and detecting gene expression. It is also a convenient means to limit the amount of modification occurring on the RNA polynucleotide because the reactive groups are spatially restricted. RNA modified at only a few 2xe2x80x2-OH positions may retain important biological functions such as serving as a template for protein translation, hybridisation and serving as a template for a polymerase.
There are many potentially useful solid phase reactants (see review by Akelah and Sherrington (1981) Chem. Rev. 81:557) such as other carboxylic acid derivatives including acid halides and acid anhydrides. Benzoyl chloride polymer bound (BCPB) heads are commercially available (Fluka, USA) with high densities of benzoyl chloride groups (approximately 2.1 mmol/g of resin). RNA can be covalently attached to the benzoyl chloride solid support via an ester linkage between the BCPB head and the 2xe2x80x2-OH group. Under appropriate reaction conditions, the 2xe2x80x2-OH groups of the RNA will react with the benzoyl chloride and become covalently coupled of the solid phase. Due to the physical separation of the benzoyl chloride groups, it is highly unlikely that all (100%) of the 2xe2x80x2-OH groups of a polynucleotide will be modified, rather only one or a few will be modified per polynucleotide. In order to immobilise RNA, it is sufficient that only one 2xe2x80x2-OH group reacts with the solid phase reagent.
Other solid phase reactive groups that may be suitable for immobilising RNA could include acid anhydrides. In the case of acid anhydrides it is important that the half of the acid anhydride attached to the solid phase is the part that reacts with the 2xe2x80x2-OH group thereby immobilising it. Otherwise, the result will be modified RNA in solution. In order to overcome this potential problem, an asymmetric acid anhyride may be attached to the solid phase so that the half of the acid anhydride attached to the solid phase reacts preferentially with the 2xe2x80x2-OH group resulting in immobilised RNA. Other examples include the use of cyclic anhydrides such as isatoic anhydride polymer bound (Cat 17344, Fluka, USA). However, it is simpler to employ carboxylic acid derivatives such as the acid halides (e.g. benzoyl chloride polymer bound), acid cyanides (e.g. benzoyl cyanide polymer bound) or acid imidazoles (e.g. N-benzoyl imidazole polymer bound) because the outcome of the reaction will be an ester linkage between the solid phase and the polynucleotide.
It will be understood that the solid phase reactant need not be limited to carboxylic acid derivatives, rather any reactant that reacts the 2xe2x80x2-OH group in a regiospecific manner is suitable. The amount of particles required to immobilise a given amount of RNA will depend on several factors such as the density of the reactive groups and the proportion of the reaction groups that will react with the 2xe2x80x2-OH. Reactive groups that are buried, for example within the resin will be unable to react with a large molecule like RNA and they will therefore be efficiently unreactive. The precise RNA binding capacity for a particular solid phase reactant will require empirical tests. If steric hindrance occurs between the polynucleotide and the reactive group due to the proximity of the solid phase a linker or spacer may be used between the solid phase and the reactive group such as xe2x80x94(CH2)12-16.
The solid phase may consist of an inorganic particle, a polymeric linear, globular or cross-linked molecule or resin. It may be made of a variety of materials or material composites such as acrylamide, agarose, cellulose, polyamide, polycarbonate, polystyrene, latex, glass and silicon. It may also possess specific properties that aid in the manipulation of the particle such as paramagnetic or magnetic properties, a diameter allowing retention by a filter, an increased density that enhances sedimentation or separation by centrifugation or incorporate a tag aiding identification, capture or quantification of the particle.
The tag may be colored, fluorescent, an isotope, an epitope for an antibody or a ligand for a protein such as streptavidin, or a protein such as an enzyme or antibody or an oligonucleotide. One purpose of the tag is to aid identification of the particle when particles from several sources are combined. For example, if mRNA from tissue 1 is immobilised on particle type A, mRNA from tissue 2 is immobilised on particle type B and particles A and B are then mixed and added to a hybridisation reaction including for example a radioactive probe. Following hybridisation, particles of type A and B are separated by means of a property unique to the tag and the radioactivity associated with each particle analysed to provide a measure of the relative gene expression in tissues 1 and 2. Other uses of the tag could include the automated quantification and validation of the number of particles in a vessel such as a 96-well plate.
The xe2x80x98particlexe2x80x99 or macromolecular structure may be incorporated into many forms such as a strip, a fibre, a matrix, a membrane, a filter, a column, a bead, a resin, a vessel wall, a pipette tip, a gel or a plate or an etched silicon device.
The reaction may be covalently attached to the solid phase using for example divinyl benzene (DVB) or associated by another means such as ionic interaction or hydrogen bonding so that in any case the RNA is firmly held to the solid phase during washing and analysis. However covalent attachment is preferred because there will be less tendency for the reactant to dissociate from the solid phase. The solid phase reactant may be incorporated into a microdevice or vessel such as those made from etched silicon and the solution containing the RNA passed over or through the region containing the reactant so that RNA immobilisation occurs in a specific region of the solid phase. This process may be automated in order to detect for example RNA viruses such as HCV and HIV in blood or body fluids. The immobilised sample might then be used for any number of down stream applications such as hybridisation, RT-PCR, TMA or NASBA.
Alternatively, the solid phase reactant may be incorporated into a blood collection device such that the RNA components of the blood are immobilised during the blood collection process. The solid phase reactant may also be coated or attached to a glass slide in many small (preferably less than 1 mm2) discrete regions. A single RNA sample from, for example a tissue source may be added (in a suitable solvent and catalyst) to one of the discrete regions thereby localising the RNA to a discrete position of the glass glide. The process may then be repeated with other RNA samples until tens, hundreds or thousands of RNA samples are attached to as many discrete regions of the slide as necessary. The slide could then be washed and subjected to hybridisation conditions with a labelled probe representing the sequence of interest such that multiple hybridisation events could be detected and with suitable instrumentation, quantitated on a single slide. Such microchip applications could have very broad applications such as diagnostic or gene expression analyses.
For certain applications it may be useful to restrict the size of the RNA that is immobilised. By using a macroporous resin with the reactant located within the pores, a size cut-off is established whereby only smaller RNA polynucleotides will be capable of accessing and therefore reacting with the reactant whilst longer RNA polynucleotides are lost. This could be useful for determining the size of the RNA polynucleotides being analysed. Alternatively it may provide a means to specifically remove short RNA polynucleotides allowing the separation of longer complete RNA polynucleotides that do not react with the solid phase. Separation of RNA based on size is currently carried out using either gel electrophoresis or column chromatography and this invention provides a simpler means to remove short polynucleotides from solution. Commercial sources of beads with defined pore sizes are available for oligonuceotide synthesis.
Following immobilisation to the particle, the RNA may be further modified at the remaining 2xe2x80x2-OH positions in order to endow it with additional desired properties such as hydrophobicity, fluorescence, radioactivity or nuclease protection. The RNA may also be used for a variety of quantitative assays such as hybridisation with labelled probes or serve as a template for RT-PCR. In the latter case, it has been found that BCPB beads have the advantage of being compatible with both the components of the reverse transcription and PCR reactions. Other purification materials such as silica beads inhibit these reactions.
A marked advantage of using PCPB beads compared with silica beads to purify RNA is that there is no elution step required to separate the RNA from the solid phase. If silica beads are added to the reverse transcription reaction they would bind both the template RNA, primers and a proportion of the enzyme leading to reduced or total inhibition of cDNA synthesis. It has been found that following RNA reaction, BCPB beads can be added directly into both the reverse transcription and PCR reactions with no such inhibition. BCPB beads may also find utility for other types of reaction such as LCR and NASBA. Adding the beads to the reaction simplifies manipulation and ensures that all the captured RNA is transferred into the reaction. The bead format could prove useful for automated systems in the diagnostic field. Thus BCPB beads are especially preferred in the present invention.
Solvents
When RNA is modified using acetic anhydride in a basic solid such as triethylamine with DMAP, the modification of the 2xe2x80x2-OH groups proceeds so quickly that base catalyst cleavage of the RNA is insignificant. However, when the reactant is attached to a solid phase such as the BCPB, it is unlikely that all the 2xe2x80x2-OH groups can be modified due to the physical immobilisation of the reactant. Therefore, even after extensive incubation times, the majority of the 2xe2x80x2-OH groups will not be modified and are therefore susceptible to cleavage by the basic solvent. It is very important that the solvent used does not lead to RNA polynucleotide cleavage. Preferred non-basic solvents include tetrahydrofuran and dimethyl sulphoxide. Neither of these solvents results in RNA cleavage yet both the RNA and catalysts such as DMAP readily dissolve in it.
It was found that some solvents increased the amount of BCPB bead sedimentation under unit gravity thereby decreasing the contact between the bead and solvent bearing the RNA. Both THF and DMF allowed the beads to be well dispersed in solution whilst water, DMSO, pyridine, triethylamine and ethyldiisopropylamine (EDPA) promoted sedimentation. This may be an effect of both density differences between the bead and solvent and hydrophobic interactions between the beads themselves. Preferred beads increase the contact between reactant and the solvent containing the RNA.
Amount and Size of Beads
It was found that adding a large excess of beads reduced the amount of RNA bound to the BCPB. For 50 ng of RNA in 1 xcexcl of water, added to 80 xcexcl of THF/DMAP (5 mg/ml) adding more than 1.5 mg of BCPB reduced the percentage of RNA bound from 74% to 48% (22 mg BCPB). It was found that during post-reaction purification of the BCPB beads that they tended to clump and stick together in aqueous solution making washing the beads difficult. This problem was overcome simply by stopping the reaction with 3 reaction volumes of 70% ethanol and briefly mixing before washing with aqueous solutions. Preferred bead sizes are those that provide a maximum surface area for reaction to occur with the RNA. Buried reactive groups will not be accessible to the RNA, therefore beads and particles with a small diameter (large surface area to volume) are preferred. However, in order to collect the beads from the liquid phase it is necessary that they are not so small that they cannot be easily pelleted by centrifugation, collected by filtration or selected by other means. Commercially available BCPB beads with a mesh size of 100-300 are readily pelleted using a centrifugal force of 1500 g for 5 sec.
Length of RNA Suitable for RT-PCR
Due to the physical separation of the reactive groups on the solid phase, a single RNA template may be linked to the solid phase via only one or a few 2xe2x80x2-sites. If the RNA temperature is relatively long and the sequence to be amplified relatively short, there will only be a small number of RNA templates that are attached within the sequence to be amplified. These templates will probably not be copied by the enzyme because the 2xe2x80x2-modification may block the passage of the enzyme. The HCV RNA genome is approximately 10,000 nucleotides long, if the PCR primers span a region of 100 bp, only 1% of the polynucleotides will not amplify due to the 2xe2x80x2-modification. However, if the RNA template is shorter, for example 1000 nucleotides and the PCR primers span 1000 bp, then none of the RNA will serve as a template for RT-PCR. It is therefore preferable to PCR amplify only short regions of the RNA. Furthermore, the reverse transcriptase primer should be as close to the 3xe2x80x2 PCR primer as possible, preferably being of identical sequence. However, for RT-PCR analysis of mRNA where the cDNA synthesis has to be primed using an oligo (dT) primer, it is preferable to use PCR primers that amplify sequences close to the 3xe2x80x2 end of the mRNA. Alternatively the RNA may be released from the solid phase using chemical or enzymatic deprotection, for example by alkali or KCN cleavage of the ester linkage before RT-PCR.
DNA Binding
Under specific conditions, some DNA will bind non-specifically to the beads probably through hydrophobic interactions with the BCPB bead. Such interactions can be reduced by using DMF, pyridine, ethyldiisopropylamine (EDPA), triethylamine or DMSO as the reaction solvent instead of THF. Non-specific DNA binding can be removed by one or more washes in 10% SDS. However, DNA may react with BCPB heads covalently via either the 3xe2x80x2 or 5xe2x80x2-OH groups. Less than 3% of a labelled DNA sample became covalently attached when DMSO was used as a solvent for the BCPB beads compared with 14% with THF as a solvent.
Protein Binding and Diagnostic Systems
In a similar manner to DNA, some protein will bind to BCPB. It was found that adding detergents to a THF solvent containing 35S-labelled cellular protein enhanced the amount of protein binding to the beads. This may be caused by the detergent unfolding the protein and therefore exposing more hydrophobic residues to the BCPB. The greatest enhancement was seen with a final concentration of 0.025% TWEEN/NP-40 detergents, THF and protein were mixed and incubated 3 min at 22xc2x0 C. with 3 mg of BCPB. Protein binding could be reduced by 50% by pre-reacting the protein with acetyl chloride. Protein binding to BCPB was not reduced if the reactivity of the benzoyl chloride group was neutralised with water prior to addition of protein. This suggests that protein binding is occurring non-specifically probably by a hydrophobic interaction.
When RNA was purified from solutions containing high concentrations of protein, it was found that a proportion of the RNA tended to bind to the beads whilst complexed with protein in a non-specific manner. Such RNA could be readily removed by several washes in 10% SDS.
It was found that highest RNA binding to BCPB beads took place when the protein and RNA were added separately to the THF solvent rather than prior to mixing with the solvent. The beads were then added last and the reaction mixed and incubated.
A practical solution to non-specific protein binding to the BCPB beads may be the use of a protein removal step before the addition of the BCPB beads to the sample. For example this may consists of a protein pre-binding step: a serum sample containing the RNA analyte may be added to, or passed through a protein binding surface such as a hydrophobic bead, membrane, filter or other protein capture surface and then the solution containing the deproteinised RNA added to the reactant-solid phase such as BCPB beads. Suitable membranes that are known to capture proteins but not nucleic acids include 0.45 xcexcm pore size IMMOBILON-P (hydrophobic polyvinylidene fluoride, Millipore, US). Alternatively, proteins could be separated from an RNA virus by filtration whereby the proteins but not the virus particles pass through an ultrafiltration membrane with a nominal molecular weight cut-off of 30 000-100 000 daltons such as CENTRICON-100, CENTRIPLUS-100 (Amicon, US). Filters with pore sizes above 0.01 xcexcm such as ISOPORE 0.05 xcexcm polycarbonate membranes (Millipore, US) should generally capture most virus particles from blood serum whilst allowing most proteins to pass through. Such collected virus particles could then be added to the reactant-solid phase such as BCPB beads.
The volume of a body fluid such as serum may be reduced prior to addition of the reactant-solid phase using a CENTRIFREE filtration device. The retained sample containing protein and viral RNA could then be added to a solvent containing the reactant-solid phase such as BCPB beads.
Blood contains approximately 70 mg/ml of protein and a large component of this is albumin and immunoglobulins. Means to reduce serum proteins include the addition of immobilised S. aureus protein A or thiophilic resin (Sigma, US) which bind immunoglobulins, a major component of blood protein. Protein A linked to a solid phase such as a bead or thiophilic resin would provide a facile means to reduce blood protein concentration.
Enhancing the Reaction
The RNA binding reaction may be enhanced by reducing the volume of the reaction to a minimum required to keep the RNA soluble and in contact with the beads. Approximately 10 xcexcl of solvent is required to immerse 1-3 mg of BCPB. It was found that adding plasmid DNA to a final concentration of 25 ng per microliter enhanced RNA binding, possibly by increasing the apparent concentration of the RNA. Other volume excluding compounds such as polyethylene glycol may also enhance RNA binding.
Addition of Detergents and Chaotropes
In order to increase specific RNA binding whilst reducing protein binding, detergents were added to a reaction containing BCPB beads in different solvents. It was found that the addition of a final concentration of 1% SDS in DMSO or THF increased the amount of RNA bound to BCPB by 33% and 42% respectively whilst 1% SDS in DMF or toluene markedly reduced RNA binding. It was also found that except for toluene, a final concentration of 1% SDS increased the amount of 35S labelled cellular protein bound to BCPB in DMF, DMSO and THF by 6.8, 2.3 and 1.85 times respectively that of a parallel reaction containing no added SDS. EDPA, pyridine or water led to low levels of RNA binding.
It was found that addition of 150 mM sodium perchlorate (final concentration) to 40 xcexcl DMSO containing 3 mg BCPB beads, 10 xcexcg BSA and 20 ng of radiolabelled RNA increased the amount of RNA bound by 2.3 fold. Even lower sodium perchlorate concentrations of 1.5 mM and 15 mM (final concentration) increased RNA binding by 1.11 and 1.33 fold respectively. This effect was not caused by the high salt concentration because a parallel reaction containing 125 M NaCl instead of sodium perchlorate bound 30% less RNA.
The addition of sodium perchlorate also increases the amount of protein bound to the beads. 40 xcexcl of DMSO containing 3 mg PCPB beads was mixed with 1 xcexcl of 35S labelled cellular protein with either 1 xcexcl of water or with 1.5 mM, 15 mM or 150 mM sodium perchlorate (final concentration); protein binding increased by 0, 1.38 and 1.5 times respectively with sodium perchlorate.
However, 600 mM urea or 400 mM guanidine thiocyanate in THF reduced BCPB reactively by 39% and 73% respectively. The addition of chaotropes and/or detergents is important because if RNA is to be isolated intact from cells, blood or other biological material, it is necessary to inhibit ribonuclease activity. One way to do this is to use a slight excess of chaotrope and/or detergent and then add this to the solid phase reactant in a suitable solvent system thereby immobilising the RNA. The solid phase may then be washed to remove contaminating proteins and DNA. Suitable chaotropes may include guanidinium chloride, guanidinium thiocyanate, sodium iodide, sodium perchlorate and sodium trichloroacetate.
An aliquot of PCPB stored at 22xc2x0 C. was opened over the course of 3 weeks and exposed to the air approximately 30 times. No reduction in reactivity was noted. BCPB is thus sufficiently stable for the present applications. Water in excess of 7.5% vol/vol in THF reduces the reactivity of BCPB. The preferred water content is 3 xcexcl or less in 40 xcexcl of THF with 1 mg of BCPB.
To reiterate, in addition to the preferred BCPB beads, the solid phase could include a particle, a bead, a membrane, agel, a slide or cover slip, an etched silicon surface, a fibre, a filter, a capillary, a tube, a vessel or a multi-vessel plate such as a 96 well plate. It will be apparent that the use of a solid phase improves handling throughput and accuracy when the RNA immobilization and analysis is automated. In this context, paramagnetic particles are favoured for their handling properties. Other preferred solid phases are those which comprise OH groups, e.g. compounds of silicon and oxygen, such as silica particles or a glass. Nucleic acid purification using silica beads is well known and widely practiced both in diagnostic and life science research. Methods of purifying RNA have been described in U.S. Pat. No. 5,234,809. Briefly the method involves binding the nucleic acid sample to silica beads in a chaotropic agent such as urea and then washing the beads in a high salt wash before it is eluted into water. The modified RNA used in the present invention has been tested for its ability to bind and be released from silica beads using a silica bead purification kit (Qiagen, Germany) and found to be particularly suited to this type of separation method (see Examples).
As mentioned above, according to U.S. Pat. No. 5,234,809, the purification of nucleic acids from biological samples involves mixing silica particles with a chaotropic salt containing the sample. Under these conditions the nucleic acid binds reversibly to the silica. During attempts to find new methods to separate modified RNA from the reaction components, it has been discovered that modified RNA will readily bind to silica beads, e.g. in the presence of 95% organic solvent. This unexpected result suggests that silica particles are suitable to bind modified RNA form a variety of organic solvents.
Agents may be employed to aid in binding the RNA to the solid phase. Bifunctional acid halides, e.g. acid chlorides, are commercially available that have effectively two reactive groups per molecule. Reagents such as sebacoyl chloride (ClOC(CH2)8COCl), adipoyl chloride (ClOC(CH2)2COCl) and glutaryl chloride (ClOC(CH2)3COCl) can be used is immobilise RNA by covalent modification of the 2xe2x80x2-OH group with consequent attachment to a solid phase. The bifunctional acid chloride can either be reacted with the RNA and then with a solid phase, or preferably with a solid phase and then the RNA sample. In both cases, the solid phase is conveniently BCPB beads, or one which has hydroxyl groups such as glass and other silicon dioxide compounds. It is known that boiling concentrated HCl exposes hydroxyl groups on the surface of glass. Such prepared glass can then be reacted with an excess of the bifunctional acid chloride, the unreacted acid chloride is removed by washing in solvent such as THF and then the RNA sampled added and allowed to react with the immobilised acid chloride, thereby becoming immobilised via the acid chloride to the glass solid phase. Bifunctional acid chlorides are preferred that have an extended hydrocarbon spacer between each end of the molecule such as with sebacoyl chloride (Cat. No. 84848 Fluka, USA) because these are likely to reduce steric hindrance between the RNA and the solid phase and therefore improve the reaction.
Modification of RNA Whilst Bound to Silica Beads
Nucleic acids become bound to the surface of silica beads, therefore tests were carried out to find out whether RNA can be modified whilst bound to the silica beads. Although it may be expected that some of the 2xe2x80x2-OH groups are protected from modification due to steric hindrance between the silica bead and the RNA, in practice, this effect was found to be undetectable leading to substantially modified RNA (see Examples). When modifying with acetic anhyride, for example, compared with an RNA modification reaction with no acetic anhydride added, the modified RNA was substantially intact and migrated identically to an acetylated RNA modified by the standard procedure. Modification, such as acetylation, of RNA can therefore occur whilst the RNA is bound or attached to a solid support or surface. This potentially offers advantages when high throughput of samples is required or when RNA is purified using silica beads for example for diagnostic purposes from blood. This is therefore a preferred aspect of the present invention.
In a further aspect, the present invention provides a kit for isolating from a sample RNA comprising an oligo- or polynucleotide, which kit comprises:
(i) a reaction system for modifying the RNA to form a modified oligo- or poly-nucleotide in which a proportion of the 2xe2x80x2-OH positions of the ribose rings bear a substituent; and
(ii) a separation system for separating material containing the substituent from the sample, so as to isolate the RNA.
Preferably, the reaction system comprises:
(a) an organic solvent; and
(b) a reactant capable of covalently modifying the 2xe2x80x2-OH position of the ribose rings of the RNA in the presence of the organic solvent. The organic solvent and reactant are discussed in further detail above.
The present invention will now be described in further detail, by way of example only, with reference to the following examples.
General Procedure
A complex mixture of DNA and RNA polynucleotides from a cellular source is treated with a lipophillic acid anhydride such as butyric anhydride (C8H14O3), pentanoic anhydride (C10H18O3), hexanoic anhydride (C12H22O3), heptanoic anhydride (C14H26O3) or benzoic anhydride (C14H10O3) as described. Longer chain lengths than heptanoic anhydride are not preferred from complete (100%) RNA modification because incomplete modification of the RNA chain has been observed, possibly due to steric hindrance between the RNA chain and the long chain anhydrides. Such reagents will modify the single terminal 3xe2x80x2-OH group and possibly the single 5xe2x80x2-OH or phosphate group of the DNA or RNA chain but all or substantially all the 2xe2x80x2-OH groups of the RNA, thereby strongly altering the physical characteristics of the RNA towards liphohilicity. However the DNA is modified at only one (3xe2x80x2-OH) or a maximum of two (one 5xe2x80x2-OH/phosphate and one 3xe2x80x2-OH) position(s) and therefore remains overall hydrophilic. This difference in lipophilicity then serves as the basis for separation of the DNA from RNA.
The hydrophobic RNA is preferably manipulated with silanised plasticware and/or with detergents such as 0.01% Tween-20, 0.01% NP40 or 0.1 Triton X-100 so that sample loss is reduced.
The capture surface or material for the modified RNA could be encapsulated within a centrifuge device such as a microcentrifuge spin-column or tube insert. Alternatively, it could be encapsulated in a pipette tip such as those commonly used to measured 1 xcexcl, 10 xcexcl, 200 xcexcl or 1 ml volumes. In either case, capture, washing and elution of the modified RNA is improved because the time required to separate the modified RNA from the contaminants is reduced.
Multiple RNA samples could be purified in parallel by the use of devices bearing multiple capture surfaces. An example would be a 96-well plate whereby each well is hydrophobic and suitable for capture, washing and elution of one modified RNA sample. A further example would be a vessel or chamber suitable for the modification reaction and in addition the capture, washing and elution of the RNA sample. In this way, both the modification and purification is carried out in the same vessel or chamber improving sample throughput and productivity. The modification, capture, washing and elution of the RNA sample could be automated and involve robots.
Method of treatment of the RNA are described in further detail in UK patent applications, Nos. 9910154.5 entitled POLYNUCLEOTIDES published 30 Jun. 1999 s GB9910154A, 9910157.8 entitled POLYNUCLEOTIDES published 30 Jun. 1999 as GB9910157A and 9910156.0 also entitled POLYNUCLEOTIDES published 30 Jun. 1999 as GB9910156A. Each of these copending patent applications was filed 30 Apr. 1999 in the name of the same applicant. For example, Example 6 of each of these copending patent applications sets out one preferred method of modifying RNA involving a DMAP-catalysed acetylation reaction. The methodology in that Example may be modified using instead of acetic anhydride, anhydrides of longer chain length including butyric or pentanoic anhydrides as set out in Example 54 of each of the copending applications. In addition, Example 1 of each of these copending patent applications sets out a method of modifying a total cellular RNA population and selection of the mRNA fraction.
It will also be apparent to those skilled in the art that a crude cellular or tissue lysate consisting of RNA, DNA, protein and lipids etc. may serve as the sample for the modification reaction. In this case, the reactant may modify not only the 2xe2x80x2-OH group of the RNA chain but also the hydroxyl bearing side chains of the amino acids tyrosine, serine or threonine of proteins. It will be apparent that such a reaction is beneficial because it will lead to the deactivation of cellular nucleases and therefore allow the selection of RNA in a more intact form. Furthermore, cells could be disrupted in the presence of the reactant so that nucleases are immediately inactivated on release from the cell and the RNA is immediately modified and therefore protected from any remaining nucleases. In this latter case, increased concentrations of the reactant in the reaction may be required in order to inactivate the nucleases fully as well as to modify fully the 2xe2x80x2-OH groups of the RNA sample.
The treated DNA and RNA sample in a high salt buffer (e.g. 1-5 M ammonium sulphate, preferably 10 mM phosphate, pH 7.0, 1.5 M ammonium sulphate) is passed over a hydrophobic column as is commonly used for reverse phased liquid chromatography. For example reversed phase packings based on silica may have bonded hydrocarbon chains of C4, C8 or C18 or other may be based on polystyrene (e.g. POROS(copyright) and OLIGO R3, PerSeptive Biosystems, USA) may be used under appropriate conditions. Further alternatives could include plastics such as polypropylene, polycarbonate, polyvinylidene fluoride (manufactured under the tradename HYDROPHOBIC DURAPORE, (Millipore, USA) and PTFE. With all plastics those with a large surface area to volume are preferred in order to increase the effective hydrophobic binding surface. These types of surfaces exist as beads of cellulose covered with butyl, octyl or phenyl groups (CELLUFINE BUTYL, CELLUFINE PHENYL and CELLUFINE OCTYL, Amicon, Mass., USA) and are well known in the art. Other bead compositions could include silica matrix modified with polyethylene- imine (SI PAE, Amicon, Mass., USA). After washing extensively with water to remove all traces of DNA, the RNA can be released by several methods. Firstly and preferably, the RNA can be deprotected by cleavage of the modifying group with 50% ammonia treatment, 10-40 mM KCN (final concentration) in 95% EtOH, K2CO3 in aqueous methanol or other conditions which are known to lead to the cleavage of the ester linkage (see Protective Groups In Organic Chemistry, 2nd edition, Ed. T. W. Greene, Wiley-Interscience). The RNA can then be collected and purified further if required by, for example oligo (dT) selection of the mRNA.
Alternatively the RNA can be removed from the solid support in its modified form using detergents such as 0.1% SDS, 0.002M TRITON X-100(trademark) (Union Carbide Chemical and Plastics, Inc.), NONIDET P-40(trademark) (Shell Oil Co.) or 1% TWEEN(trademark) (ICI Americas, Inc.) in aqueous solution. Or alternatively by lowering the salt concentration (preferably 10 mM phosphate, pH 7.0) in the binding buffer to a point where elution is brought about. Another approach is the addition of a chaotropic agent such as ethylene glycol, urea, guanidine-HCl or thiocyanate salts. Solvents such as isopropanol, methanol, ethanol, DMSO or acetronitrile may also be used to elute the RNA up to a final concentration of 40% (v/v) in water.
The invention will now be described in further detail, by way of example only, with reference to the following Examples and the accompanying drawings, in which: